Japanese Encephalitis (JE) Vaccine in Vero cell can be easily purified by zonal centrifugation at non continuous sucrose gradients (36% and 60%), 32 600g for 4h. The calf serum protein and other nonviral proteins in the vaccine were almost separated from the JE virus. The residual calf serum protein was less than 0.5μg/mL, and the total protein was less than 30 μg/mL. The residual Vero cell DNA in the vaccine was less than 100 pg/0.5mL. The titer of purified Japanese Encephalitis vaccine is six times higher than China control vaccine. This method is recommended as an available method to purify JE vaccine from Vero cell in large-scale because it is simple, rapid and inexpensive.